As illustrated in FIG. 1, angiogenesis, the induction of new blood vessels, is critical for normal growth as well as pathogenesis of various disorders. In cancers, angiogenesis accelerates growth of solid tumors and provides a gateway to metastasis via the newly formed vasculature. In contrast, therapeutic angiogenesis is important for reducing the effects of tissue ischemia and preventing organ failure. The process of angiogenesis is tightly controlled by a number of specific mitogens, among which vascular endothelial growth factor (VEGF) and its receptors play a key role. The levels of VEGF are upregulated across a broad range of tumors, and play a causal role in oncogenic signaling. In cells and tissues, transcription of VEGF gene is regulated by hypoxia-inducible factors. Among them, Hypoxia-Inducible Factor 1 (“HIF-1”) is the main regulator of oxygen-dependent transcription in a majority of organs and accounts for the increase in expression of hypoxia-inducible genes. HIF-1 consists of an oxygen-sensitive a and a constitutively expressed β subunit. Under well-oxygenated conditions, HIF-1α is hydroxylated (Ivan et al., “HIFα Targeted for VHL-mediated Destruction by Proline Hydroxylation: Implications for O2 Sensing,” Science 292:464-8 (2001)), ubiquitinated, and degraded by the ubiquitin—proteasome system. Under hypoxia, HIF-1α is stabilized and translocates into the nucleus where heterodimerization with its constitutively expressed binding partner, aryl hydrocarbon receptor nuclear translocator (“ARNT”) (Wood et al., “The Role of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) in Hypoxic Induction of Gene Expression,” J. Biol. Chem. 271:15117-23 (1996)) results in binding to a cognate hypoxia response element (“HRE”) (Forsythe et al., “Activation of Vascular Endothelial Growth Factor Gene Transcription by Hypoxia-inducible Factor 1,” Mol. Cell. Biol. 16:4604-13 (1996)). The heterodimer then recruits transcriptional coactivators, p300, CBP, and SRC-1, resulting in the upregulation of the hypoxia-inducible genes. Regulation of the activity of hypoxia-inducible factors includes three critical steps: (i) inhibition of hydroxylation of two proline residues to preclude interaction of HIF-1α with pVHL, a part of ubiquitin ligase complex, thereby preventing its proteasomal destruction; (ii) inhibition of hydroxylation of Asn803 by Factor Inhibiting HIF-1α (“FIH”) (Lando et al., “FIH-1 Is an Asparaginyl Hydroxylase Enzyme That Regulates the Transcriptional Activity of Hypoxia-inducible Factor,” Genes & Develop. 16:1466-71 (2002)) to enable recruitment of coactivators, which trigger overexpression of hypoxia inducible genes, including genes encoding angiogenic peptides such as VEGF and VEGF receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), as well as proteins involved in altered energy metabolism, such as the glucose transporters GLUT1 and GLUT3, and hexokinases 1 and 2 (Forsythe et al., “Activation of Vascular Endothelial Growth Factor Gene Transcription by Hypoxia-inducible Factor 1,” Mol. Cell. Biol. 16:4604-13 (1996); Okino et al., “Hypoxia-inducible Mammalian Gene Expression Analyzed in Vivo at a TATA-driven Promoter and at an Initiator-driven Promoter,” J. Biol. Chem. 273:23837-43 (1998)); and (iii) interaction of promoter-bound HIF-1α/1β with coactivator protein p300 (or the homologous CREB binding protein, CBP) leading to upregulation of transcription.
The interaction between the cysteine-histidine rich 1 domain (“CH1”) of p300/CBP and the C-terminal transactivation domain (“C-TAD786-826”) of HIF-1α (Freedman et al., “Structural Basis for Recruitment of CBP/p300 by Hypoxia-inducible Factor-1α,” Proc. Nat'l Acad. Sci. USA 99:5367-72 (2002); Dames et al., “Structural Basis for Hif-1α/CBP Recognition in the Cellular Hypoxic Response,” Proc. Nat'l Acad. Sci. USA 99:5271-6 (2002)) mediates transactivation of hypoxia-inducible genes (Hirota & Semenza, “Regulation of Angiogenesis by Hypoxia-inducible Factor 1,” Crit. Rev. Oncol. Hematol. 59:15-26 (2006); Semenza, “Targeting HIF-1 for Cancer Therapy,”Nat. Rev. Cancer 3:721-32 (2003)) (see FIG. 2A). As illustrated in FIGS. 2A-C, structural studies provide a molecular basis for this transcription factor-coactivator interaction and identify two short α-helical domains from HIF-1α as key determinants for its recognition by p300 (Freedman et al., “Structural Basis for Recruitment of CBP/p300 by Hypoxia-Inducible Factor-1α,” Proc. Nat'l Acad. Sci. USA 99:5367-72 (2002); Dames et al., “Structural Basis for Hif-1α/CBP Recognition in the Cellular Hypoxic Response,” Proc. Nat'l Acad. Sci. USA. 99:5271-76 (2002)). Synthetic mimics of these domains could inhibit HIF-1α/p300 or HIF-1α/CBP complex formation and regulate transcription. Key residues contributing to the binding of one of the two helices (PDB code IL8C, residues 139-147) are shown in FIG. 2C.
Because interaction of HIF-1α C-TAD with transcriptional coactivator p300/CBP is a point of significant amplification in transcriptional response, its disruption with designed protein ligands could be an effective means of suppressing aerobic glycolysis and angiogenesis (i.e., the formation of new blood vessels) in cancers (Hirota & Semenza, “Regulation of Angiogenesis by Hypoxia-inducible Factor 1,” Crit. Rev. Oncol. Hematol. 59:15-26 (2006); Rarnanathan et al., “Perturbational Profiling of a Cell-line Model of Tumorigenesis by Using Metabolic Measurements,” Proc. Nat'l Acad. Sci. USA 102:5992-7 (2005); Underiner et al., “Development of Vascular Endothelial Growth Factor Receptor (VEGFR) Kinase Inhibitors as Anti-angiogenic Agents in Cancer Therapy,” Curr. Med. Chem. 11:731-45 (2004)). Although the contact surface of the HIF-1α C-TAD with p300/CBP is extensive (3393 Å2), the inhibition of this protein—protein interaction may not require direct interference. Instead, the induction of a structural change to one of the binding partners (p300/CBP) may be sufficient to disrupt the complex (Kung et al., “Small Molecule Blockade of Transcriptional Coactivation of the Hypoxia-inducible Factor Pathway,” Cancer Cell 6:33-43 (2004)).
Although inhibition of nuclear protein—protein interactions with small molecules in the past has proven to be difficult (Arkin & Wells, “Small-molecule Inhibitors of Protein—Protein Interactions: Progressing Towards the Dream,” Nat. Rev. Drug Discov. 3:301-17 (2004)), screens for high-affinity protein ligands have resulted in several remarkable accomplishments (Kung et al., “Small Molecule Blockade of Transcriptional Coactivation of the Hypoxia-inducible Factor Pathway,” Cancer Cell 6:33-43 (2004); Issaeva et al., “Small Molecule RITA Binds to p53, Blocks p53-HDM-2 Interaction and Activates p53 Function in Tumors,” Nat. Med. 10:1321-8 (2004); Lepourcelet et al., “Small-molecule Antagonists of the Oncogenic Tcf/β-Catenin Protein Complex,” Cancer Cell 5:91-102 (2004); Vassilev et al., “In Vivo Activation of the p53 Pathway by Small-molecule Antagonists of MDM2,” Science 303:844-8 (2004); Grasberger et al., “Discovery and Cocrystal Structure of Benzodiazepinedione HDM2 Antagonists That Activate p53 in Cells,” J. Med. Chem. 48:909-12 (2005); Ding et al., “Structure-based Design of Potent Non-peptide MDM2 Inhibitors,”J. Am. Chem. Soc. 127:10130-1 (2005); Berg et al., “Small-molecule Antagonists of Myc/Max Dimerization Inhibit Myc-induced Transformation of Chicken Embryo Fibroblasts,”Proc. Nat'l Acad. Sci. USA 99:3830-5 (2002); International Patent Publication No. WO 2006/066775 to De Munari et al.). Two small molecules, chaetocin 1 (Hauser et al., “Isolation and Structure Elucidation of Chaetocin,” Helv. Chim. Acta 53(5):1061-73 (1970)) and chetomin 2 (Waksman & Bugie, “Chaetomin, a New Antibiotic Substance Produced by Chaetomium Cochliodes I. Formation and Properties,” J. Bacteriol. 48:527-30 (1944)), have been shown to inhibit the interaction between HIF-1α C-TAD and p300/CBP and to attenuate hypoxia-inducible transcription. Despite the initial encouraging reports, further design of inhibitors of the HIF-1 pathway is needed, because both 1 and 2 have induced coagulative necrosis, anemia, and leukocytosis in experimental animals. It would be desirable to identify other inhibitors of the HIF-1 pathway that lack or have diminished side effects.
The present invention is directed to overcoming these and other deficiencies in the art.